Role of fibronectin in platelet adhesion and aggregation impqct of biomechanics and β3 integrin on fibrillogenesis

i Role of fibronectin in platelet adhesion DQGDJJUHJDWLRQLPSDFWRIELRPHFKDQLFVDQGȕ integrin on fibrillogenesis Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Khon C Huynh aus Ho Chi Minh/Vietnam Düsseldorf, Oktober 2012 ii aus dem Institut für Hämostaseologie, Hämotherapie und Transfusionsmedizin der Heinrich-Heine Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Herr Prof Dr Rudiger E Scharf Korreferent: Herr Prof Dr Dieter Willbold Tag der mündlichen Prüfung: 06.11.2012 iii Acknowledgement The writing of this dissertation is one of the most academic challenges I have ever had to face It would not have been completed without the supports and efforts of many kind people around me I own my deepest gratitude to them First of all, I would like to express my gratitude to my advisor Prof Dr Rudiger E Scharf who had given me the chance to pursue my studies at the IHHTM Working in his Institute has been fun, meaningful and supportive I am also thankful to him for giving me the freedom to explore my own, and many chances to visit scientific conferences to enlarge my knowledge in the field of Hemostasis and Thrombosis His patience and support helped me to improve my ability to write scientific reports and manuscripts My deepest gratitude is to my topic supervisor and my Biotruct project principal investigator Dr Volker R Stoldt His advices, support and friendship are invaluable on both a scientific and a personal level He has been always there listening, giving advices and providing support despite of his enormous work pressures His insightful comments and constructive criticism at different stages of my research were thought-provoking and helped me to focus my ideas Without his support, my life would not have been started smoothly in Germany which is a foreign country for me I would like to take this opportunity to thank Prof Dr Dieter Willbold who is also my cosupervisor I am indebted to him for his patience, encouragement, and network support as well as for reading, commenting on my reports and my views of science A very special thank goes to Dr Marianne Gyenes and Dr Abdelouahid Elkhattouti for being my best colleagues and friends over all these years My life in the lab would not have been so joyful and efficient without their friendship and support Thanks to them for sharing with me and helping me to solve the difficulties in scientific and personal life Together, we had many nice times visiting conferences, travelling that would be one of the most unforgettable moments in my graduate student time I would like to acknowledge the financial, academic and technical support of the NRW graduate school Biostruct, particularly Dr Christian Dumpitak and Dr Cordula Kruse for coordinating this project I am also thankful to Prof Dr Margitta Elvers for reading and correcting my thesis, Elisabeth Kirchhoff and Bianka Masen-Weingardt for their experiences and supports in experiments with Fn purification, platelet isolation, platelet aggregation I am also grateful to the former or current iv members and students (MD and PhD) at IHHTM, for their various forms of support during my study Many friends have helped me to stay sane through these difficult years Their support and care helped me to overcome problems and to stay focused on my graduate study I greatly value their friendship Finally, my wife Pham, Thi Luc Hoa together with our family have supported and helped me along the course of this dissertation by giving encouragement and providing the endless love, support and strength For any errors or inadequacies that may remain in this work, of course, the responsibility is entirely my own v Content Introduction 1.1 Fibronectin (Fn) 1.1.1 Structure of Fn 1.1.2 Plasma Fn and cellular Fn 1.1.3 Major steps in Fn assembly 1 1.2 Integrins 1.2.1 Fn receptors (integrins) on the platelet surface 1.2.2 Integrin activation 1.2.3 Fn-integrin interaction during fibril assembly 1.3 Fn in platelet functions in hemostasis 1.3.1 Fn in platelet adhesion 1.3.2 Fn in platelet aggregation 1.3.3 Fn assembly in platelet adhesion and aggregation 8 1.4 Description and importance of the present studie Materials and Methods 10 11 2.1 Materials 2.1.1 General equipment and kits 2.1.2 General chemicals and materials 2.1.3 Antibodies, ligands and fluorescence dyes 2.1.4 Other materials 2.1.5 Buffer and SDS-PAGE gel compositions 11 11 11 12 12 12 2.2 Methods 2.2.1 Isolation of plasma Fn 2.2.2 Platelet preparation 2.2.3 Platelet aggregation assay 2.2.4 Platelet adhesion assay 2.2.5 Fn labeling for FRET (Fluorescence resonance energy transfer) 2.2.6 Sensitivity of FRET to changes in Fn conformation 2.2.7 Fn unfolding by platelets monitored by FRET 2.2.8 DOC-solubility assay to study Fn assembly by adherent platelets under flow conditions 2.2.9 Statistical analysis 13 13 13 13 14 14 14 15 15 16 Results 17 3.1 Purification of Fn from human plasma 17 3.2 Fn enhances platelets adhesion but decreases platelet aggregation 18 vi 3.2.1 3.2.2 3.3 Fn decreases platelet aggregation Fn enhances platelet adhesion Sensitivity of FRET to conformational changes of Fn in denaturing conditions 18 18 20 3.4 FRET analyses of Fn unfolding by platelets under static conditions 3.4.1 Adherent but not suspended platelets progressively unfold Fn during interaction 3.4.2 ȕLQWHJULQ-dependent unfolding of Fn during platelet adhesion under static conditions 3.4.3 Effect of actin polymerization on Fn unfolding by adherent platelets under static conditions 22 22 23 24 3.5 Biomechanical stress modulates Fn unfolding by adherent platelets 3.5.1 Fn assembly by adherent platelets under flow conditions 3.5.2 (IIHFWVRIȕLQWHJULQDQWLERGLHVRQ)QXQIROGLQJE\DGKHUHQWSODWHOHWVXQGHUIORZFRQGLWLRQV 26 26 27 Discussion 29 4.1 Purification of plasma Fn 29 4.2 Dual role of Fn in platelet adhesion and aggregation 30 4.3 Functions of Fn in association with its unfolding and assembly 31 4.4 Factors affect unfolding of Fn by adherent platelets 4.4.1 ȕLQWHJULQ-dependent Fn unfolding by adherent platelets under static condition 4.4.2 Fn unfolding by adherent platelet can be modulated by cytoskeleton drugs 4.4.3 Acceleration of Fn assembly by adherent platelets by shear stress under flow 4.4.4 5ROHRIȕLQWHJULQVĮ,,EȕDQGĮYȕ XQGHUIORZFRQGLWLRQV Conclusions and perspectives Summary References Appendix 34 34 35 36 37 38 vii Abbreviations ADP Adenosine diphosphate APS Ammonium persulphate BSA Bovine serum albumin CaCl Calcium chloride Cyto D Cytochalasin D DOC Deoxycholate EDTA Ethylenediaminetetraacetic acid Fg Fibrinogen Fn Fibronectin FRET Fluorescence resonance energy transfer GdnHCl Guanidine hydrochloride H2O water Jas Jasplakinolide KCl Potassium chloride kDa kilo Dalton KH PO Monopotassium phosphate Lat A Latrunculin A MgCl Magnesium chloride Na HPO Sodium phosphate dibasic NaCl Sodium chloride NaN Sodium azide PBS Phosphate buffered saline PHSRN Proline-histidine-serine-arginine-asparagine sequence PMA Phorbol 12-myristate 13-acetate PMMA para-Methoxy-N-methylamphetamine PMSF Phenylmethanesulfonyl fluoride viii PRP Platelet-rich plasma Reopro Abciximab antibody RGD Arginine-glycine-aspartic acid sequence SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TEMED N, N, N', N'-tetramethylethylenediamine UV Ultraviolet vWF von Willebrand factor % Percentage °C degree Celsius μg microgram μM micromolar g gram (weight) L Liter M Molar (= mol/L) mg milligram ml milliliter nm nanometer rpm revolutions per minute s-1 inverse seconds 1 Introduction 1.1 Fibronectin (Fn) 1.1.1 Structure of Fn Fn is a dimeric glycoprotein of 230-270 KDa subunits that is present in the extracellular matrix and in blood plasma [4-5] Fn is a modular protein that comprises three types of repeating units: twelve type I repeats (FnI), two type II repeats (FnII) and 15-17 type III repeats (FnIII) [4, 6] (Figure 1.1) The type I and type II repeats contain two intramolecular disulfide bonds to stabilize their folded structure while the type III repeat is a 7-VWUDQGHGȕ-barrel structure lacking disulfide bonds [7-9] Therefore, the type III repeats can undergo conformational changes [10] Sets of modules are organized into functional domains including the N-terminal 70 kDa domain, the 120 kDa central binding domain and the heparin-binding domain [1, 11] The diverse set of binding domains allows Fn to interact with multiple cellular integrin receptors, collagen, gelatin (but not in vivo), heparin and other extracellular molecules including Fn itself [3] The primary gene transcript of Fn can generate multiple mRNA transcript leading to distinct Fn isoforms by alternatively splicing [11] There are about 20 monomeric isoforms in humans and about 12 isoforms in rodents and cows [12] Alternatively splicing occurs at three sites amongs the type III repeats: extra type III domains EIIIA/EDA (between III11 and III12), EIIIB/EDB (between III7 an III8) and the V region/IIICS (between III14 and III15) [3] Each of these splicing regions may carry out some unique functions of Fn regarding cell adhesive activities or protein solubility and stability ACCEPTED MANUSCRIPT 19 surfaces This oppositional effect can be explained by the fact that only adherent platelets can unfold and assemble FN Folded FN may act as a competitor for the binding sites of FG or other PT plasma proteins on platelets in suspension, while the unfolded form can be a bridge to connect RI platelets-ligands and promote platelet-to-platelet interaction, thereby enhancing adhesion and SC thrombus formation (Figure 6) There are two models of conformational changes of FN that can be detected by FRET NU measurement: compact-to-extended conformation (arm separation) without loss of secondary MA structure or compact-to-unfolded conformation changes (26) Due to the experimental design, the observed decrease of about 6% in FRET signals in the present study does not provide any D information about the exact conformational changes in the FN molecule However, based on TE studies reporting the detection of FN fibrils on adherent platelets, it can be noted that the observed conformational changes is not simply a process of opening arms of FN Indeed, it is a AC CE P compact-to-extended process in the modules of FN which is required for FN fibril formation It has been reported that adherent platelets on different thrombogenic surfaces show different abilities in assembling FN fibrils on their surfaces (47) Here, we demonstrate that FN assembly is also dependent on the adhesion of platelets Signaling molecules regulating actin polymerization play an essential role in controlling FN unfolding and assembly by fibroblasts (19) In addition, Src, a tyrosine kinase, plays a crucial role in integrin-dependent outside-in signaling (32) Our results demonstrate that platelets adherent onto FN have a significantly higher activity of pY418 Src than platelets in suspension under all applied conditions The difference in Src phosphorylation between adherent platelets and suspended platelets can also observed in experiments with immobilized FG or soluble FG, respectively (M Gyenes, unpublished data) This difference in Src phosphorylation between ACCEPTED MANUSCRIPT 20 suspended and adherent platelets is significantly reduced after adding PP1 but not apyrase to platelets Hence, the increase in Src phosphorylation is dependent on the adhesion of platelets PT onto immobilized ligand A previous report suggests that Src is required to initiate cytoskeletal RI events which are important for generating the required biomechanical forces to unfold FN (32) of adherent platelets to unfold and assemble FN SC Thus, we conclude that a high level of Src phosphorylation is one of the intracellular mechanism NU In summary, our studies show that FN plays a dual role in haemostasis Using fluorescence MA techniques and DOC-solubility assays, we demonstrate that differences in structural changes of FN, when interacting with suspended and adherent platelets, may explain its dual role in platelet D adhesion and aggregation Adherent platelets can unfold and hence, induce FN fibril assembly to TE enhance adhesion, whereas FN cannot be unfolded by suspended platelets and therefore, reduces platelet aggregation This contention is supported by quantitative differences in Src signaling AC CE P seen in resuspended and adherent platelets Acknowledgements: We thank Drs Kristopher E Kubow and Maria Mitsi for their experiences in fibronectin purification and labeling We thank Elisabeth Kirchhoff and Bianka MaassenWeingart for their assistances in platelet aggregation assays Sources of Funding: This work was supported by DFG (Sonderforschungsbereich 612 Teilprojekt B2) and the NRW Research School BioStruct, by grants from the Ministry of Innovation, Science, Research and Technology of the German Federal State North RhineWestphalia (NRW) and from the 'Gründerstiftung zur Förderung von Forschung und wissenschaftlichen Nachwuchs an der Heinrich-Heine-Universität Düsseldorf Disclosures: None ACCEPTED MANUSCRIPT 21 References AC CE P TE D MA NU SC RI PT Mosher DF Fibronectin San Diego: Academic Press; 1989 xviii, 474 p p Hynes RO Fibronectins New York: Springer-Verlag; 1990 xv, 546 p p Mosher DF Fibronectin Prog Hemost Thromb 1980;5:111-51 PubMed PMID: 6999530 Epub 1980/01/01 eng Clark RA Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin J Invest Dermatol 1990 Jun;94(6 Suppl):128S-34S PubMed PMID: 2161886 Epub 1990/06/01 eng Singer AJ, Clark RA Cutaneous wound healing N Engl J Med 1999 Sep 2;341(10):73846 PubMed PMID: 10471461 Epub 1999/09/02 eng Ni H, Yuen PS, Papalia JM, Trevithick JE, Sakai T, Fassler R, et al Plasma fibronectin promotes thrombus growth and stability in injured arterioles Proc Natl Acad Sci U S A 2003 Mar 4;100(5):2415-9 PubMed PMID: 12606706 Pubmed Central PMCID: 151355 Epub 2003/02/28 eng Bastida E, Escolar G, Ordinas A, Sixma JJ Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces Blood 1987 Nov;70(5):1437-42 PubMed PMID: 3663940 Epub 1987/11/01 eng Moon DG, Kaplan JE, Mazurkewicz JE The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation Blood 1986 Feb;67(2):450-7 PubMed PMID: 3942831 Epub 1986/02/01 eng Santoro SA Inhibition of platelet aggregation by fibronectin Biochem Biophys Res Commun 1983 Oct 14;116(1):135-40 PubMed PMID: 6416252 Epub 1983/10/14 eng 10 Dixit VM, Haverstick DM, O'Rourke K, Hennessy SW, Broekelmann TJ, McDonald JA, et al Inhibition of platelet aggregation by a monoclonal antibody against human fibronectin Proc Natl Acad Sci U S A 1985 Jun;82(11):3844-8 PubMed PMID: 2582428 Pubmed Central PMCID: 397884 Epub 1985/06/01 eng 11 Thurlow PJ, Kenneally DA, Connellan JM The role of fibronectin in platelet aggregation Br J Haematol 1990 Aug;75(4):549-56 PubMed PMID: 2207006 Epub 1990/08/01 eng 12 Reheman A, Yang H, Zhu G, Jin W, He F, Spring CM, et al Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor Blood 2009 Feb 19;113(8):1809-17 PubMed PMID: 19036705 Epub 2008/11/28 eng 13 Salmeron-Sanchez M, Rico P, Moratal D, Lee TT, Schwarzbauer JE, Garcia AJ Role of material-driven fibronectin fibrillogenesis in cell differentiation Biomaterials 2011 Mar;32(8):2099-105 PubMed PMID: 21185593 Epub 2010/12/28 eng 14 Johnson KJ, Sage H, Briscoe G, Erickson HP The compact conformation of fibronectin is determined by intramolecular ionic interactions J Biol Chem 1999 May 28;274(22):15473-9 PubMed PMID: 10336438 Epub 1999/05/21 eng 15 Sottile J, Mosher DF Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules Biochemistry 1993 Feb 16;32(6):1641-7 PubMed PMID: 8431444 Epub 1993/02/16 eng 16 Schwarzbauer JE, Sechler JL Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly Curr Opin Cell Biol 1999 Oct;11(5):622-7 PubMed PMID: 10508649 Epub 1999/10/06 eng 17 Sechler JL, Takada Y, Schwarzbauer JE Altered rate of fibronectin matrix assembly by deletion of the first type III repeats J Cell Biol 1996 Jul;134(2):573-83 PubMed PMID: 8707839 Pubmed Central PMCID: 2120877 Epub 1996/07/01 eng ACCEPTED MANUSCRIPT 22 AC CE P TE D MA NU SC RI PT 18 Cho J, Mosher DF Role of fibronectin assembly in platelet thrombus formation J Thromb Haemost 2006 Jul;4(7):1461-9 PubMed PMID: 16839338 Epub 2006/07/15 eng 19 Geiger B, Bershadsky A, Pankov R, Yamada KM Transmembrane crosstalk between the extracellular matrix cytoskeleton crosstalk Nat Rev Mol Cell Biol 2001 Nov;2(11):793-805 PubMed PMID: 11715046 Epub 2001/11/21 eng 20 Wu C, Keivens VM, O'Toole TE, McDonald JA, Ginsberg MH Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix Cell 1995 Dec 1;83(5):715-24 PubMed PMID: 8521488 Epub 1995/12/01 eng 21 Zhong C, Chrzanowska-Wodnicka M, Brown J, Shaub A, Belkin AM, Burridge K Rhomediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly J Cell Biol 1998 Apr 20;141(2):539-51 PubMed PMID: 9548730 Pubmed Central PMCID: 2148448 Epub 1998/05/23 eng 22 Guan JL, Shalloway D Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation Nature 1992 Aug 20;358(6388):690-2 PubMed PMID: 1379699 Epub 1992/08/20 eng 23 Wary KK, Mainiero F, Isakoff SJ, Marcantonio EE, Giancotti FG The adaptor protein Shc couples a class of integrins to the control of cell cycle progression Cell 1996 Nov 15;87(4):733-43 PubMed PMID: 8929541 Epub 1996/11/15 eng 24 Huveneers S, Danen EH The interaction of SRC kinase with beta3 integrin tails: a potential therapeutic target in thrombosis and cancer ScientificWorldJournal 2010;10:1100-5 PubMed PMID: 20563533 Epub 2010/06/22 eng 25 Engvall E, Ruoslahti E Binding of soluble form of fibroblast surface protein, fibronectin, to collagen Int J Cancer 1977 Jul 15;20(1):1-5 PubMed PMID: 903179 Epub 1977/07/15 eng 26 Smith ML, Gourdon D, Little WC, Kubow KE, Eguiluz RA, Luna-Morris S, et al Forceinduced unfolding of fibronectin in the extracellular matrix of living cells PLoS Biol 2007 Oct 2;5(10):e268 PubMed PMID: 17914904 Pubmed Central PMCID: 1994993 Epub 2007/10/05 eng 27 Olorundare OE, Peyruchaud O, Albrecht RM, Mosher DF Assembly of a fibronectin matrix by adherent platelets stimulated by lysophosphatidic acid and other agonists Blood 2001 Jul 1;98(1):117-24 PubMed PMID: 11418470 Epub 2001/06/22 eng 28 Mosher DF, Williams EM Fibronectin concentration is decreased in plasma of severely ill patients with disseminated intravascular coagulation J Lab Clin Med 1978 May;91(5):729-35 PubMed PMID: 641397 Epub 1978/05/01 eng 29 Plow EF, Ginsberg MH Specific and saturable binding of plasma fibronectin to thrombin-stimulated human platelets J Biol Chem 1981 Sep 25;256(18):9477-82 PubMed PMID: 6270087 Epub 1981/09/25 eng 30 Schwarzbauer JE Identification of the fibronectin sequences required for assembly of a fibrillar matrix J Cell Biol 1991 Jun;113(6):1463-73 PubMed PMID: 2045422 Pubmed Central PMCID: 2289042 Epub 1991/06/01 eng 31 Litvinov RI, Nagaswami C, Vilaire G, Shuman H, Bennett JS, Weisel JW Functional and structural correlations of individual alphaIIbbeta3 molecules Blood 2004 Dec 15;104(13):397985 PubMed PMID: 15319287 Epub 2004/08/21 eng 32 Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, et al Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton J Cell Biol 2002 Apr 15;157(2):265-75 PubMed PMID: 11940607 Pubmed Central PMCID: 2199242 Epub 2002/04/10 eng ACCEPTED MANUSCRIPT 23 AC CE P TE D MA NU SC RI PT 33 Matuskova J, Chauhan AK, Cambien B, Astrof S, Dole VS, Piffath CL, et al Decreased plasma fibronectin leads to delayed thrombus growth in injured arterioles Arterioscler Thromb Vasc Biol 2006 Jun;26(6):1391-6 PubMed PMID: 16528004 Epub 2006/03/11 eng 34 Maurer LM, Tomasini-Johansson BR, Mosher DF Emerging roles of fibronectin in thrombosis Thromb Res 2010 Apr;125(4):287-91 PubMed PMID: 20116835 Pubmed Central PMCID: 2877496 Epub 2010/02/02 eng 35 Nievelstein PF, D'Alessio PA, Sixma JJ Fibronectin in platelet adhesion to human collagen types I and III Use of nonfibrillar and fibrillar collagen in flowing blood studies Arteriosclerosis 1988 Mar-Apr;8(2):200-6 PubMed PMID: 3348760 Epub 1988/03/01 eng 36 Sochynsky RA, Sykes B, University of Oxford Medical Sciences Division Collagen and fibronectin in platelet function [Thesis (D Phil )]: University of Oxford, 1980.; 1980 37 To WS, Midwood KS Plasma and cellular fibronectin: distinct and independent functions during tissue repair Fibrogenesis Tissue Repair 2011;4:21 PubMed PMID: 21923916 Pubmed Central PMCID: 3182887 Epub 2011/09/20 eng 38 Dixit VM, Haverstick DM, O'Rourke KM, Hennessy SW, Grant GA, Santoro SA, et al A monoclonal antibody against human thrombospondin inhibits platelet aggregation Proc Natl Acad Sci U S A 1985 May;82(10):3472-6 PubMed PMID: 2582413 Pubmed Central PMCID: 397798 Epub 1985/05/01 eng 39 Wang Y, Reheman A, Spring CM, Kalantari J, Marshall AH, Wolberg AS, et al Plasma fibronectin supports hemostasis and regulates thrombosis J Clin Invest 2014 Oct;124(10):428193 PubMed PMID: 25180602 Pubmed Central PMCID: PMC4191008 Epub 2014/09/03 eng 40 Cho J, Mosher DF Impact of fibronectin assembly on platelet thrombus formation in response to type I collagen and von Willebrand factor Blood 2006 Oct 1;108(7):2229-36 PubMed PMID: 16735600 Pubmed Central PMCID: 1895571 Epub 2006/06/01 eng 41 Erickson HP, Carrell NA Fibronectin in extended and compact conformations Electron microscopy and sedimentation analysis J Biol Chem 1983 Dec 10;258(23):14539-44 PubMed PMID: 6643500 Epub 1983/12/10 eng 42 Rocco M, Carson M, Hantgan R, McDonagh J, Hermans J Dependence of the shape of the plasma fibronectin molecule on solvent composition Ionic strength and glycerol content J Biol Chem 1983 Dec 10;258(23):14545-9 PubMed PMID: 6643501 Epub 1983/12/10 eng 43 Williams EC, Janmey PA, Ferry JD, Mosher DF Conformational states of fibronectin Effects of pH, ionic strength, and collagen binding J Biol Chem 1982 Dec 25;257(24):14973-8 PubMed PMID: 7174679 Epub 1982/12/25 eng 44 Tanabe J, Fujita H, Iwamatsu A, Mohri H, Ohkubo T Fibronectin inhibits platelet aggregation independently of RGD sequence J Biol Chem 1993 Dec 25;268(36):27143-7 PubMed PMID: 8262952 Epub 1993/12/25 eng 45 Leng L, Kashiwagi H, Ren XD, Shattil SJ RhoA and the function of platelet integrin alphaIIbbeta3 Blood 1998 Jun 1;91(11):4206-15 PubMed PMID: 9596668 Epub 1998/05/30 eng 46 Baugh L, Vogel V Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer J Biomed Mater Res A 2004 Jun 1;69(3):525-34 PubMed PMID: 15127399 Epub 2004/05/06 eng 47 Somers CE, Mosher DF Protein kinase C modulation of fibronectin matrix assembly J Biol Chem 1993 Oct 25;268(30):22277-80 PubMed PMID: 8226736 Epub 1993/10/25 eng ACCEPTED MANUSCRIPT 24 Figure legends Figure Dual effect of FN on platelet adhesion and aggregation PT Washed platelets were stimulated with 10 µg/mL collagen (A) or 40 nM PMA (B) in the absence RI or presence of 300 µg/mL plasma FN Platelets aggregation was measured by using SC aggregometer Resting platelets did not aggregate without treatment with agonist (data not shown) NU 200 µL of CMFDA-labeled platelets (5ì107/mL) without or with 300 àg/mL plasma FN in MA HEPES Tyrode’s buffer containing mM CaCl2 were placed on collagen- (C) or FN-coated wells (D) of 96-well plate and incubated for 30 at 37°C In parallel experiments, platelets were D allowed to adhere onto immobilized ligand in the presence of 10 µM ADP Adhesion was TE quantified by fluorescence intensity of CMFDA as described in “Materials and methods” Values AC CE P represent the mean ± SD of three individual experiments (*) p< 0.05 Figure Fluorescence conjugation and characterization of labeled FN (A) Schematic sketch of the putative positions of fluorophores conjugated to FN molecule for FRET FN was doubly labeled with AF 488 (donor) and AF546 (acceptor) as described in “Materials and methods” (B) Schematic sketch of FN conformations in GdnHCl solutions correlated with FRET FN in solution of M GdnHCl is in a compact structure so that it exhibits highest FRET signal As GdnHCl concentration in solution increases to > M or > 4M causing FN to partially unfolded or unfolded, respectively, FRET signal decreases (C) Fluorescence emission spectra of doubly labeled FN exposed to solutions with increasing concentrations of GdnHCl Spectra have been normalized to the donor peak so that changes in FRET are indicated solely by changes in the acceptor peak ACCEPTED MANUSCRIPT 25 (D) Reference curve probing FN unfolding in GdnHCl solutions Labeled FN was exposed to 0-4 M GdnHCl and fluorescence intensities of donors and acceptors were recorded FRET PT was calculated as ratio of IA/ID FRET of FN in M GdnHCl solution was shown as RI 100% SC Figure FN unfolding and assembly by platelets monitored by FRET analysis and DOC- NU solubility assay MA (A) FN unfolding by suspended and adherent platelets monitored by FRET Labeled FN was mixed with at least 10 fold excess of unlabeled FN to prevent energy transfer between D adjacent protein molecules FN mixtures (10 µg/mL) were incubated for h at room TE temperature with washed platelets in suspension or with platelets adherent onto immobilized FN (50 µg/mL) In both settings, platelets were stimulated by 40 nM PMA AC CE P For control, FRET signals of FN mixtures without platelets were recorded (B) Representative fluorescence emission spectra of labeled FN incubated with adherent platelets over h Spectra have been normalized to the donor peak so that changes in FRET are indicated solely by changes in the acceptor peak (C) & (D) Fluorescence measurement of deposited FN fibrils on platelets Platelets in suspension or adherent onto immobilized FN were incubated with FN488 (60 µg/mL) for 1-3 h in the presence of ADP (C) or PMA (D) DOC-solubility assays were performed to compared the amount of FN fibrils deposition on platelet surfaces as described in “Materials and Methods” Values represent the mean ± SD of three individual experiments (*) p